Arthur C. Huen M.D., Ph.D, is currently completing training as a dermatologist committed to pursuing basic research investigations into wound healing. Intrigued by research from the beginning, he had pursued training in basic science research during medical school, resulting in doctorate training focusing on the role of intercellular adhesion, specifically the role of desmoplakin, a desmosomal plaque protein, in providing a strong state of intercellular adhesion via its connection to intermediate filaments. This work was completed at Northwestern University under the mentorship of Kathleen J. Green Ph.D (Mayberry Professor of Pathology, Professor of Dermatology, Associate Chairman of Pathology, Northwestern University). His current clinical training is being pursued at the University of Pittsburgh Medical Center (UPMC) Department of Dermatology and his research training is being conducted in the laboratory of Alan Wells M.D., D.M.S (Vice Chairman, Department of Pathology, Thomas J Gill III Professor of Pathology and Medical Director of Section of Laboratory Medicine at the University of Pittsburgh Medical Center) and under the supervisory and career mentorship of Louis Falo (Professor and Chairman, Department of Dermatology, UPMC). Both Departments are fully committed to Dr. Huen's development and have designed a transitional training situation that focuses on investigative experience, combined with some classroom and seminar/short course training in targeted areas. The main thrust will be to introduce Dr. Huen to the research community as a junior investigator. The Well's laboratory studies molecular mechanisms regulating cellular migration in both physiologic and pathologic processes such as wound healing and tumor invasion, respectively. The focus is on developing new hypotheses derived from discovery approaches in the literature and laboratory to solve complex questions involving cell-tissue communications. The major techniques utilized in the laboratory, and in which Dr. Huen is being trained, include tissue culture and organotypic culture as well as microscopy. Much of the in vitro work is verified using mouse models, with animals being housed at the nearby Pittsburgh VA Medical Center. A unique aspect of this work is the focus on ex vivo organotypic models that are specifically engineered to allow the molecular manipulations of in vitro in an model approaching the organ complexity of in vivo. In addition, there are several other laboratories on the same floor and interactions are frequent via combined weekly lab meetings as well as semimonthly formal conferences. Training within this environment and funding of the currently proposed research plan will provide a transition for Arthur Huen to become an independent medical scientist. The currently proposed project focuses on the role of chemokine receptor CXCR3 ligands, including IP9 (CXCL11, ITAC) and IP10 (CXCL10), in coordinating the transition of cutaneous wound healing from the proliferative phase to the remodeling phase. In vitro studies have demonstrated that stimulation with IP9 results in increased migration by keratinocytes whereas stimulation in fibroblasts results in decreased migration. The goal of this research proposal is to determine the signals responsible for initiating the cascade of CXCR3 ligands that starts the remodeling phase of cutaneous wound healing. It is hypothesized that keratinocytes near, but distal to the wound edge that are undergoing re-differentiation during re-epithelialization are responsible for expression of CXCR3 ligands IP9 and, to a lesser extent, IP10. Involvement of these re- differentiating keratinocytes would best fit the spatio-temporal expression of IP9 and IP10. To test this hypothesis, three specific aims are proposed. The first aim will examine the role of cellular confluence, which induces keratinocyte differentiation, on expression of chemokines IP9 and IP10. In the second aim, the hypothesis that re-establishment of intercellular junctions by keratinocytes is a trigger for chemokine expression will be examined. Lastly, the third aim will examine the effect of exogenous IP9 and IP10 stimulation on the rate of re-epithelialization in organotypic skin culture and mouse skin. These studies will be accomplished not only in routine tissue culture, but also in animals using unique genetically engineered mouse models, and in novel ex vivo organotypic cultures systems. Part of this proposal will involve establishing and using an all-human engineered skin organ culture in which the different components can be individually altered at the molecular level. This will then be a unique and highly useful tool for launching the initial independent projects of Dr. Huen. In addition, successful completion of these studies will establish an innovative model of wound healing for further explorations some of which would allow for translational development. PUBLIC HEALTH RELEVANCE: The proposed research project will study control mechanisms for signals released by cells near the edge of skin wounds that initiate the later stages of wound healing. Errors in the timing or the amount of signal released by these cells may result abnormal wound healing; for example, lack of signaling seems to slow the rate of skin wounds to close while too much signaling is expected to close wounds quickly but form weaker, depressed scars. Understanding the control of these signals may lead to better ways to treat chronic wounds and to more rapidly restore skin function and appearance after trauma.